1,721,053 research outputs found
Chemical exchange in proteins : relaxation of multiple- and single-quantum coherences studied by NMR
No full textNuclear Magnetic Resonance (NMR) spectroscopy offers many ways to investigate dynamic properties of molecules. A wide variety of experimental techniques can probe molecular dynamics on time scales that range from 10-12 to 103 seconds. Many lines of evidence point to the existence of large scale dynamics on milli- to microsecond time scale that are essential to molecular function. Those conformational motions can give rise to chemical exchange effects. So far in the literature two methods have been mainly used to study such processes: CPMG echo trains and R1ρ spin-lock relaxation experiments. The former is suited to investigate processes on the millisecond time scale, whereas R1ρ can probe processes ranging from milliseconds down to ca. microseconds. CPMG and R1ρ studies of the relaxation rates of single quantum (SQ) coherences can be complemented by multiple quantum coherence (MQ) coherences experiments which can provide information whether two (or more) spins are affected simultaneously by chemical exchange. The goal of this thesis is to characterize chemical exchange in proteins by new (Heteronuclear Double Resonance, HDR) and existing (relaxation compensated CPMG) methods. The presence of chemical exchange has been identified in APO-rMUP using the classical CPMG experiment on single quantum 15N magnetization, whereas in HOLO-rMUP no presence of chemical exchange has been found. Most of the thesis is dedicated to the analysis and the application of a new method, HDR, which is designed to determine the cross-relaxation rates between MQ operators. In fact, in heteronuclear systems, correlated chemical exchange contributes to cross-relaxation between MQ coherences 2IxSx and 2IySy. This method, based on MLEV-32 and WALTZ-16 used in double resonance mode, is designed to effectively preserve all relevant MQ coherences simultaneously so that the interconversion between MQ operators can only arise through cross-relaxation. This is an essential requirement if we want to measure, for example, small cross-relaxation rates between MQ operators. We have treated the effects of coherent evolution during the HDR sequences by numerical simulations and Average Hamiltonian Theory (AHT) showing that, under most conditions, the dynamics of MQ coherences is not affected by coherent evolution. This result is proved experimentally on the 15N-1H pair of selectively deuterated tBoc-glycine. The effect of relaxation during HDR sequences leads to an effective relaxation superoperator which is explored by numerical simulations and predicted by Average Liouvillian Theory (ALT). Finally the HDR MLEV-32 sequence is applied to amide 1H-15N pairs in ubiquitin and KIX proteins. In the case of ubiquitin the HDR MLEV-32 experiments and the numerical simulations run with parameters obtained from the literature are in good agreement, suggesting that HDR sequences could be used as a tool to obtain information about the dynamics of the system.LRM
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
The conformational space and dynamics of the partially disordered transcription factor engrailed-2 explored with magnetic resonance
No full textLes protéines intrinsèquement désordonnées (IDP), dépourvues d’une structure rigide et stable, constituent une classe de protéines diverses et fonctionnellement importantes. La résonance magnétique nucléaire (RMN) est une technique spectroscopique bien établie pour caractériser les propriétés conformationnelles et dynamiques des IDP avec une résolution atomique. L’espace conformationnel, en général large et varié, des IPD en fait une cible difficile pour la biologie structurale dont le but est de déterminer avec précision et exactitude les propriétés structurales, dynamique et physico-chimiques qui sous-tendent la fonction des macromolécules biologiques. Ce manuscrit présente une étude biophysique détaillée de la région intrinsèquement désordonnée (IDR) du facteur de transcription Engrailed-2, avant tout par RMN. Après une présentation de cette homéoprotéine, nous décrivons les protocoles d’expression et de purification de cette protéine isotopiquement marquée. Nous introduisons ensuite une nouvelle approche pour la caractérisation des mouvements pico- et nanoseconde des protéines intrinsèquement désordonnées à partir de données de relaxation des spins nucléaires enregistrées à plusieurs champs magnétiques. Les effets de relaxation paramagnétique (PRE) ont été utilisés pour identifier des interactions transitoires entre la région désordonnée et l’homéodomaine d’Engrailed-2. L’interaction d’Engrailed-2 avec l’ADN a été étudiée en détail en utilisant l’anisotropie de fluorescence sur une série de constructions de la protéine, afin de mettre en lumière le rôle de la partie désordonnée dans l’interaction avec l’ADN. Nous avons également employé la résonance paramagnétique électronique pour tenter de détecter une interaction potentielle entre le noyau hydrophobe de l’hexapeptide dans la région désordonnée et l’homéodomaine. Les couplages dipolaires résiduels (RDC) dans les paires 1H-15N, Cα-Hα et Cα-C′ ont également été mesurés sur des échantillons d’Engrailed en milieu anisotrope. Ces données seront essentielles pour reconstituer l’espace conformationnel d’Engrailed 2. L’ensemble des approches présentées a permis de constituer un socle solide de connaissances qui permettent de mieux comprendre les propriétés conformationnelles, dynamiques et fonctionnelles de l’IDR d’Engrailed-2.Intrinsically Disordered Proteins (IDPs), which lack a stable rigid structure constitute a large and functionally important class of proteins. Nuclear Magnetic Resonance (NMR) is a well-established technique to characterize the structural and dynamical features of IDPs at atomic resolution. The broad conformational space of IDPs makes them challenging targets for structural biology to define their precise structural features and motions, the physical and chemical properties that underlie their biological functions. The present thesis establishes biophysical investigation of the disordered region of the transcription factor Engrailed-2 (13.5 kDa) primarily by NMR. After describing the protocol of expression and purification of the isotopically labeled protein, we present a novel approach to characterize the pico – nano second motions in IDPs using nuclear spin relaxation data at multiple fields. Paramagnetic Relaxation Enhancements (PREs) are used to identify transient long-range interactions between the disordered region and the folded homeodomain of Engrailed-2. Binding to DNA was studied by fluorescence anisotropy and highlights the role of the disordered region in the DNA binding. We used Electron Paramagnetic Resonance (EPR) to probe the potential interaction between the hydrophobic cluster (hexapeptide) in the disordered region and the homeodomain. The one-bond 1H-15N, Cα-Hα and Cα-C′ residual dipolar couplings (RDCs) measured for Engrailed-2 provide important constraints for the refinement of the conformational space of Engrailed_2. All these approaches provide valuable insights in understanding the structural, dynamical and functional properties of this IDP
Relaxation par RMN multi-champs dans les biomolécules
No full textLa relaxation des spins nucléaires est un phénomène fondamental en Résonance Magnétique Nucléaire (RMN). Au cours d’une expérience, elle conduit à des pertes de polarisation affectant la qualité des spectres. Afin de développer de nouvelles séquences d’impulsion, il est essentiel de prendre en compte ses effets, voire de les optimiser, comme dans le cas des expériences de type TROSY (Transverse Relaxation Optimized SpectroscopY). Après une brève introduction à la théorie de la relaxation en phase liquide, nous détaillons comment cette théorie a été implémentée dans le but de calculer efficacement les vitesses de relaxation d’un grand nombre de systèmes de spins. La théorie de la relaxation nous a permis de comprendre le spectre de groupes méthyl dans la protéine Ubiquitine, et enregistré avec une évolution zéro quantum à bas champs et une détection à haut champs en utilisant un spectromètre RMN à deux champs. Cela nous a conduits à étendre le champ d’application de la théorie du methyl-TROSY. Par ailleurs, nous avons introduit le concept de TROSY à deux-champs. Il repose non seulement sur la sélection d’opérateur de spin ayant des propriétés de relaxation favorables, mais également sur la sélection adéquate des champs magnétiques pour l’évolution sous l’effet du déplacement chimique tout en conservant la sensibilité des hauts champs pour la détection. La mesure des vitesses de relaxation, constitue un outil de choix pour la caractérisation de la dynamique sur des échelles de temps allant de la pico- à la seconde, et plus. Nous présentons ici des outils pour analyser la dépendance en champs magnétique de vitesses de relaxation enregistrées sur une large gamme de champs magnétiques. Enfin, nous présentons quelques modèles de mouvements prenant en compte la nature des mouvements dans les protéines. En particulier, nous montrons l’existence d’un mécanisme de relaxation associé à des différences de CSA (Chemical Shift Anisotropy) dans les chaînes latérales aliphatiques.Nuclear spin relaxation is a fundamental phenomenon in Nuclear Magnetic Resonance (NMR). During the course of an experiment, it leads to polarization losses that can be detrimental to the spectrum quality. Taking spin relaxation into account when developing NMR pulse sequences appears essential, and can reveal itself beneficial, as shown in TRansverse Optimized SpectroscopY (TROSY) type of experiments. After a brief introduction to nuclear spin relaxation theory in liquid, we will detail how it has been implemented to efficiently compute relaxation rates of arbitrary spin systems. Nuclear spin relaxation theory has been used to understand the spectrum of methyl groups in the protein Ubiquitin recorded with zero-quantum evolution at low field and signal detection at high field using a two-field NMR spectrometer. This led us to extend the methyl-TROSY theory beyond its original conditions of application. In addition, we introduced the concept of two-field TROSY which relies not only on the selection of spin quantum operators with favorable relaxation properties, but also on the proper selection of the magnetic field for chemical shift labeling while retaining high-field high-sensitivity detection. Relaxation measurements report on dynamic properties over timescales ranging from pico- to seconds and more is unique. Here, we present tools to analyze the field-dependence of relaxation rates recorded while moving the sample inside the bore of the spectrometer to extend the range of available magnetic fields. Finally, we discuss models of motions adapted to the nature of internal motions in protons. We reveal the existence of a rotamer Chemical Shift Anisotropy (CSA) dependent relaxation mechanism in aliphatic side-chains
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Field-cycling long-lived-state NMR of 15N2 spin pairs
No full textA range of nuclear magnetic resonance spectroscopy and imaging applications are limited by the short lifetimes of magnetisation in solution. Long-lived states, which are slowly relaxing configurations of nuclear spins, have been shown to alleviate this limitation. Long-lived states have decay lifetimes significantly exceeding the longitudinal relaxation time , in some cases by an order of magnitude. Here we present an experimental case of a long-lived state for a 15N labelled molecular system in solution. We observe a strongly biexponential decay for the long-lived state, with the lifetime of the slowly relaxing component exceeding 40 minutes, ∼21 times longer than the spin-lattice relaxation time . The lifetime of the long-lived state was revealed by using a dedicated two-field NMR spectrometer capable of fast sample shuttling between high and low magnetic fields, and the application of a resonant radiofrequency field at low magnetic field. The relaxation characteristics of the long-lived state are examined
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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